Effects Of Ionizing Radiation On Ultraweak Bioluminescence Of Human Hepatoma HepG2 Cells

The malignant tumor is the main disease which is harmful to human, and the primary therapeutic tools are chemotherapy, radiotherapy and surgery. Radiotherapy has been about a hundred years of history, in China there are about 70% of the malignant tumor patients need to receive radiation therapy. Different tumors from different sources and their organizations, the degree of tumor cell differentiation and proliferation of different levels, and its response to ionizing radiation there is great difference, that is, a different tumor, the radiosensitivity is different. Radiosensitivity of tumors is the development of tumor radiation therapy program, select the primary tumor based on radiation dose. But is still a lack of detection of tumor cell radiation sensitivity of the methods and indicators.Ultraweak bioluminescence of cells provided a new chance for solving the problem. All living organisms can emit a very weak photon emission,which is called ultraweak bioluminescence. Living cells'ultraweak bioluminescence is the signals from the cells which carry many informations about oxidative metabolism and proliferation activities. Tumor cells have their own characteristic ultraweak bioluminescence signal, when they being subjected to the external ionizing radiation, the cells'ultraweak bioluminescence certainly will change their intensity and features. So we can get the informations of tumor cells'radiosensitivity from ultraweak bioluminescence.OBJECTIVE: To study the biological effects of ionizing radiation on HepG2 cells and their ultraweak boiluminescence changes.METHODS: Exponential growth phase of human hepatoma carcinoma HepG2 cells were irradiated with ⁶⁰Coγ-rays, the absorbed dose were: 0.5, 1, 2, 4, 6, 8 Gy, to non-irradiated HepG2 cells (0Gy) for control, after irradiation the tumor cells on the following observation at 48 hours:(1) Using light microscope and electron microscopy to observe cell morphology changes in the structure and ultrastructure;(2) Application of MTT colorimetric test for detection of cell proliferation activity;(3) Measured the changes of cell cycle and apoptosis rate by Flow CytoMetry;(4) Using immunohistochemical methods to detect cell p27 protein expression and the change of distribution;(5) The application of low-permeability, chromosome diffusion and Giemsa staining cells to detect G2-chromosome aberrations;(6) The use of flat-panel experimental determination of colony-forming cells after irradiation survival fraction of clone;(7) The changes of cell ultraweak bioluminescence intensity was examined by IFFM-D flow chemiluminescence analyzer.RESULTS:(1) Distortion of cell morphology under a light microscope, including a huge body, the more obvious the greater the dose; electron microscopy shows that the integrity of cell membrane destruction, collapsing envelope, karyopycnosis, hypersegmentation of nucleus, cytoplasmic vacuoles found.(2) MTT colorimetric test results showed that cell growth inhibition rate and the absorbed dose was positively correlated, r = 0.962(P<0.01).(3) Cell mitosis was delay after exposure, in 48 hours after irradiation 2Gy, G₂/M phase block happened, at 4Gy when in the highest proportion of G2 phase cells, and a clear apoptotic peak. After more than 4Gy dose the proportion of G₂ phase cells no longer increased, but the proportion of apoptotic cells at 6Gy reach a maximum of 30.5%.(4) Cells after irradiation, p27 protein expression was significantly inhibited, and mainly distributed in the cytoplasm outside the nucleus.(5) Exposure to cell G₂-chromosome aberrations and absorbed dose has significant relevance, the correlation coefficient r = 0.937(P<0.01), that is, the higher the absorbed dose, the more chromosomal aberrations.(6) Colony-forming cell test showed that by survival fraction after irradiation with the absorbed dose by the scores in line with the linear-square model (lnS=-αD-βD²)fitting that in the low-dose irradiation, the cells with the lethal dose is directly proportional to distortion, the cell survival curve is linear ; in the high-dose irradiation, the fatal dose of distortion is proportional to the square.(7) Cells by 48 hours after radiation, the ultraweak bioluminescence intensity and the absorbed dose was a negative correlation, correlation coefficient r =-0.803(P<0.01); Ultraweak luminescence intensity and cell clone survival fraction of the correlation coefficient r=0.841(P<0.01), note luminous intensity and cell survival fraction was significantly positively correlated.CONCLUSIONS:(1) Changes of HepG2 cells membrane structure induced byγ-rays lead to changes of cells'functions and forms,which resulted in the cell cycle extension,growth inhibition,cell survival fraction decreased, chromosome aberration which were correlated with exposure dose(P<0.01). P27 protein expression was inhibited and distribution was changed ,the cell death in principal was apoptosis at low dose and necrosis at high dose.(2) Ultraweak bioluminescence intensity of HepG2 cells was correlated with both exposure dose and cell survival fraction(P<0.01), ultraweak bioluminescence intensity could reflect radiosensitivity of tumor cells. It is limited that colony forming assay expend too much time what is used to measure cells radiosensitivity. Ultraweak chemiluminescence detection technology is expected to become a fast and accurate new indicator to predict of radiation sensitivity of tumor cells.